Examining the effects of immune mediators on the vasculature of the blood and lymphatic systems of the human skin: IL-1β, TNFα and CpG oligodeoxynucleotides

Abstract

The induction of endothelial cytokines and the regulation of endothelial vascular permeability are essential for cellular based immunity through lymphatic intravasation of activated immune cells to lymphocyte extravasation at the site of inflammation. While the appreciation of dermal endothelial cells and their role in innate and adaptive immunity is increasing, the mechanisms are not yet fully understood or clinically exploited. The purpose of this study is to assess the influence of IL-1β, TNFα and CpG oligodeoxynucleotides on the vasculature of the blood and lymphatic systems of the human skin. The study has exploited two distinct but complementary experimental model systems; one involved in vitro culturing of human microvascular endothelial (HMEC-1) cells, and the other involved ex vivo culturing of human dermal explants. Extensive analysis of cytokine secretion by HMEC-1 cells in response to stimuli demonstrated different profiles; both IL-1β and TNFα induced increased secretion of IL-8, MCP-1, RANTES, sICAM-1, sVCAM-1, and VEGF, whereas IL-1β further induced IL-6, MIP1α, MIP1β, G-CSF, and GM-CSF and consistently demonstrated greater potency. Interestingly, on their own, CpG oligodeoxynucleotides did not influence cytokine secretion. However, co-stimulation with IL-1β and CpG oligodeoxynucleotides enhanced MCP-1 secretion and attenuated IL-8 secretion, in comparison with IL-1β stimulation alone. TEER measurements of HMEC-1 cells demonstrated a robust and rapid decrease in TEER following IL-1β stimulation, whilst TNFα demonstrated a later onset but equally potent decrease. Stimulation with CpG ODN2006 and CpG ODN2395 initiated a slow continual decrease in TEER, whilst CpG ODN2216 resulted in a rapid and sustained decrease. Preliminary immunohistochemistry studies of ex vivo human dermal explants indicated that intradermal injection with IL-1β or TNFα does not substantially influence the expression levels of the endothelial markers; PECAM-1 (CD31), LYVE-1, VE-cadherin (CD144), or Claudin-5. Conversely, a marked downregulation of PECAM-1, VE-cadherin, and Claudin-5 was observed following intradermal injection with any one of the three CpG oligodeoxynucleotides. These findings suggest that the inflammatory conditions induced by IL-1β and TNFα have different underlying mechanisms, while the contrasting conditions induced by CpG oligodeoxynucleotides indicate a more selective and distinct role in inflammation. Further studies may elucidate this role and help to identify clinical opportunities.